Analyte sensors and methods of using same

ABSTRACT

Provided are sensors for determining the concentration of an analyte in a sample fluid. In certain embodiments, the sensors include conductive particles and exhibit improved uniformity of distribution of one or more sensing chemistry components, increased effective working electrode surface area, and/or reduced entry of interfering components into a sample chamber of the sensor. Methods of using and manufacturing the sensors are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/360,120, filed Nov. 23, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/949,996, filed Jul. 24, 2013, issued as U.S.Pat. No. 9,535,027, which claims priority based on U.S. ProvisionalApplication No. 61/675,696, filed Jul. 25, 2012, the disclosure of whichis incorporated by reference herein in its entirety.

INTRODUCTION

In many instances it is desirable or necessary to regularly monitor theconcentration of particular constituents in a fluid. A number of systemsare available that analyze the constituents of bodily fluids such asblood, urine and saliva. Examples of such systems conveniently monitorthe level of particular medically significant fluid constituents, suchas, for example, cholesterol, ketones, vitamins, proteins, and variousmetabolites or blood sugars, such as glucose. Diagnosis and managementof patients suffering from diabetes mellitus, a disorder of the pancreaswhere insufficient production of insulin prevents normal regulation ofblood sugar levels, requires carefully monitoring of blood glucoselevels on a daily basis. A number of systems that allow individuals toeasily monitor their blood glucose are currently available. Such systemsinclude electrochemical biosensors, including those that comprise aglucose sensor that is adapted to determine the concentration of ananalyte in a bodily fluid (e.g., blood) sample.

A person may obtain a blood sample by withdrawing blood from a bloodsource in his or her body, such as a vein, using a needle and syringe,for example, or by lancing a portion of his or her skin, using a lancingdevice, for example, to make blood available external to the skin, toobtain the necessary sample volume for in vitro testing. The person maythen apply the fresh blood sample to a test strip, whereupon suitabledetection methods, such as colorimetric, electrochemical, or photometricdetection methods, for example, may be used to determine the person'sactual blood glucose level.

Analyte sensors with improved performance, such as increased accuracyand response times, are desirable. The present disclosure providessensors, and methods of using such sensors, meeting these and a varietyof other needs.

SUMMARY

Provided are sensors for determining the concentration of an analyte ina sample fluid. In certain embodiments, the sensors include conductiveparticles and exhibit improved uniformity of distribution of one or moresensing chemistry components, increased effective working electrodesurface area, and/or reduced entry of interfering components into asample chamber of the sensor. Methods of using and manufacturing thesensors are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show a schematic view of non-uniform reagent distributionupon drying of a detection reagent solution in the sample chamber of asensor.

FIGS. 2A-2E show a schematic view of substantially uniform reagentdistribution upon drying of a particle-containing detection reagentsolution in the sample chamber of a sensor.

FIG. 3 is a schematic view of a first embodiment of a sensor strip inaccordance with the present disclosure.

FIG. 4 is an exploded view of the sensor strip shown in FIG. 3, thelayers illustrated individually with the electrodes in a firstconfiguration.

FIG. 5 is an exploded view of a second embodiment of a sensor strip inaccordance with the present disclosure, the layers illustratedindividually with the electrodes in a second configuration.

FIGS. 6A and 6B are microscopic images depicting an approximatemonolayer of conductive particles disposed on a working electrodesurface.

FIG. 7 is a graph showing the peak currents and response times ofanalyte sensors having no conductive particles or various numbers oflayers of conductive particles in their sample chambers.

FIG. 8 is a graph showing the average currents (n=1 to 3) for differentAu-coated microsphere loadings.

DETAILED DESCRIPTION

Provided are sensors for determining the concentration of an analyte ina sample fluid. In certain embodiments, the sensors include conductiveparticles and exhibit improved uniformity of distribution of one or moresensing chemistry components, increased effective working electrodesurface area, and/or reduced entry of interfering components into asample chamber of the sensor. Methods of using and manufacturing thesensors are also provided.

Before the sensors and methods of the present disclosure are describedin greater detail, it is to be understood that the sensors and methodsare not limited to particular embodiments described, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the sensors andmethods will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the sensors and methods. The upperand lower limits of these smaller ranges may independently be includedin the smaller ranges and are also encompassed within the sensors andmethods, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe sensors and methods.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods belong. Although any methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the sensors and methods, representativeillustrative sensors, methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the sensors, methods and/or materials in connection with whichthe publications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present sensors and methods are not entitled toantedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the sensors and methods,which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the sensors and methods, which are, forbrevity, described in the context of a single embodiment, may also beprovided separately or in any suitable sub-combination. All combinationsof the embodiments are specifically embraced by the present inventionand are disclosed herein just as if each and every combination wasindividually and explicitly disclosed, to the extent that suchcombinations embrace operable processes and/or devices/systems/kits. Inaddition, all sub-combinations listed in the embodiments describing suchvariables are also specifically embraced by the present sensors andmethods and are disclosed herein just as if each and every suchsub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentsensors and methods. Any recited method can be carried out in the orderof events recited or in any other order which is logically possible.

Sensors

Disclosed herein are sensors for determining the concentration of ananalyte in a sample fluid. In certain embodiments, a sensor havingparticulate matter disposed on an area of a working electrode of thesensor is provided. For example, a sensor may have conductive particlesdisposed on all or a portion of a working electrode within a samplechamber of the sensor. In certain aspects, the particulate matter isprovided on the surface of a working electrode as part of a compositionthat includes at least one analyte detection reagent (e.g., ananalyte-responsive enzyme and/or a redox mediator) that facilitatedetection of an analyte. That is, a composition (e.g., a suspension)that includes, e.g., conductive particles, an analyte-responsive enzymeand/or a redox mediator, may be disposed on all or a portion of aworking electrode surface of the sensor. In other embodiments, theparticulate matter may first be disposed on a surface of a workingelectrode, and a composition that includes one or more detectionreagents, e.g., an analyte-responsive enzyme and/or a redox mediator, issubsequently disposed on the conductive particles.

In each of the sensor embodiments of the present disclosure, theconductive particles may be configured and arranged to provide for moreeven distribution of the sensing reagents on the surface of the workingelectrode as compared to the distribution absent the particles, increasethe effective surface area of the working electrode, and/or prevent theentry of interfering substances (e.g., red blood cells) into a samplechamber of the sensor.

Sensors Having Improved Uniformity of Distribution of One or MoreAnalyte Detection Reagents

Embodiments of the present disclosure relate to sensors having improveduniformity of distribution of one or more analyte detection reagents byinclusion of particulate matter (e.g., microparticles), where thedetection reagent and particulate matter are disposed on a surface of aworking electrode of the sensor, such as in vitro or in vivo analytesensors. For example, embodiments of the present disclosure provide forinclusion of microparticles in a solution, such as a detection reagentsolution, resulting in more uniform distribution of the detectingreagent after the detection reagent solution is dried on the surface ofthe working electrode. Also provided are methods of manufacturing theanalyte sensors and methods of using the analyte sensors in analytemonitoring.

In certain aspects, during the manufacturing process for the subjectanalyte sensors, a detection reagent solution is contacted with asurface of a substrate (e.g., a surface of a working electrode), forminga deposition of the solution on the surface of the substrate. In somecases, the solution is allowed to dry and cure. One difficulty withproducing analyte sensors in which the reagent solution is deposited ina channel (e.g., a channel constituting a sample chamber region definedby a spacer layer of the sensor) is that—as the reagent solutiondries—the reagent tends to deposit preferentially at the sides of thechannel. In such instances, the reagent will not be uniformlydistributed across the channel (e.g., along the axis perpendicular tothe flow of a sample into the sample chamber), and the center of thechannel may be partially denuded of reagent, resulting in the analytenot reacting completely in the center of the channel, particularly athigh analyte concentrations (e.g., high blood glucose concentrations).This non-uniformity may adversely affect performance characteristics ofthe sensor, such as decreasing response times and/or accuracy ascompared to a sensor having substantially uniform distribution of thedetection reagent on the working electrode surface.

The process of non-uniform reagent distribution as a reagent solutiondries in a sample chamber of a sensor is schematically illustrated inFIGS. 1A-1E. A portion of the partially assembled sensor includes samplechamber 102 defined by substrate 104 and spacer layer 106. As shown inFIGS. 1A-1D, as reagent solution 110 dries, the reagent (shown as dotsin reagent solution 110) preferentially deposits at sides 108 of thesample chamber. FIG. 1E shows a top view of the portion of the sensorshown in FIG. 1D.

Embodiments of the present disclosure are based on the discovery thatthe addition of particulate matter (e.g., carbon nanopowder,microparticles (e.g., conductive microspheres), and/or the like) to areagent solution used in the manufacture of in vitro or in vivo analytesensors improves uniformity and/or distribution of one or more detectionreagents (e.g., an analyte-responsive enzyme and/or redox mediator) on asurface of the sensor. The principle of adding particulate matter to areagent solution for improved reagent uniformity and distribution isschematically illustrated in FIGS. 2A-2E. As shown, a portion of apartially manufactured sensor includes sample chamber 202 defined bysubstrate 204 (which may be a working electrode surface) and spacerlayer 206. Reagent solution 210 is disposed in sample chamber 202 andincludes detection reagent(s) (shown as dots in reagent solution 210)and particulate matter (shown as circles in reagent solution 210). Asshown in FIGS. 2A-2D, as reagent solution 210 dries, the particulatematter inhibits the preferential deposition of reagent components atsides 208 of the sample chamber. As shown in FIG. 2D, the result is adry reagent-particulate matter composition disposed on the substrate(e.g., a working electrode surface) in the sample chamber region, thecomposition having substantially uniform distribution of the detectionreagent(s) attributable to the particulate matter being present in thereagent solution during the drying. FIG. 2E shows a top view of theportion of the sensor shown in FIG. 2D.

For ease of illustration, the area of the sample chamber consumed by theparticulate matter in FIGS. 2A-2E is substantial. It will be appreciatedthat the particle density of the reagent solution/suspension may beadjusted to achieve a desired amount/configuration of particles in thesample chamber upon drying of the solution/suspension. For example, theparticle density may be chosen to achieve uniform distribution of theanalyte detection reagent, and also to provide less than a single layerof particles on the surface of the substrate. In other aspects, theparticle density may be chosen to achieve uniform distribution of theanalyte detection reagent, and also to provide a monolayer, bilayer, ormore layers of the particles on the surface of the substrate.

Accordingly, in certain embodiments, the present disclosure providessensors for determining the concentration of an analyte (e.g., glucose,a ketone, etc.) in a sample fluid. The sensors include a first substratehaving a proximal end and a distal end, the first substrate defining afirst side edge and a second side edge of the sensor extending from theproximal end to the distal end of the first substrate, the distal endbeing configured and arranged for insertion into a sensor reader.According to this aspect, the sensors also include a second substratedisposed over the first substrate, a working electrode disposed on oneof the first and second substrates, a counter electrode disposed on oneof the first and second substrates, and a spacer disposed between thefirst and second substrates and defining a sample chamber that comprisesthe working electrode and the counter electrode. Also according to thisaspect, the sensors include a dry composition disposed on an area of theworking electrode, the composition comprising an analyte detectionreagent and conductive particles configured and arranged to provide forsubstantially uniform distribution of the detection reagent on the areaof the working electrode.

By “substantially uniform distribution” of the detection reagent ismeant that if the area of the working electrode upon which the drycomposition is disposed was subdivided into equal sized smaller areas(or “sub-areas”), these sub-areas would each have the same orsubstantially the same quantity of disposed analyte detection reagent,within the uncertainty of the measurement method used to quantify thedisposed reagent. In certain embodiments, the quantity of analytedetection reagent disposed on the sub-areas does not differ betweensub-areas by more than about 25%. For example, the quantity of detectionreagent disposed on the sub-areas does not differ between sub-areas bymore than about 20%, 15%, 10%, 5%, 2%, or more than about 1%.

For example, a rectangular working electrode (with, e.g., a depositedenzyme and an osmium-based mediator) of width 1.5 mm and length 4 mm,could be carefully scribed into 3 smaller rectangular sub-areas, each0.5 mm by 4 mm. Each sub-area could then be washed with buffer toextract the deposited enzyme and mediator into a known volume of liquid,e.g., about 1 mL. Enzyme activity assays could then be used to quantifythe amount of enzyme in each sub-area, and/or atomic absorptionspectroscopy (e.g., for osmium) could be used to quantify the amount ofmediator.

The area of the working electrode on which the composition is disposedmay comprise any desired amount of conductive particles. In certainaspects, the area of the working electrode on which the dry compositionis disposed includes between about 10² and 10¹⁰ conductiveparticles/mm². For example, the area of the working electrode mayinclude between about 10³ and 10⁹ conductive particles/mm², betweenabout 10⁴ and 10⁸ conductive particles/mm², between about 10⁴ and 10⁷conductive particles/mm², or between about 10⁵ and 10⁶ conductiveparticles/mm². The particle density may depend, e.g., on the diameter ofthe particles, the concentration of the particles in the “wet”composition applied to the working electrode surface prior to drying,and/or the like.

According to certain embodiments, the sensors having substantiallyuniform distribution of the detection reagent on the area of the workingelectrode are in vitro analyte sensors. For example, the sensors may bein vitro analyte test strips. Such analyte sensors may have any desiredconfiguration. For example, the sensors may be “tip fill” sensors, wherethe sample fluid is contacted at an aperture positioned at the proximaltip of the sensor for introducing the sample fluid into the samplechamber of the sensor. Alternatively, the sensors may be “side fill”sensors, where the sample fluid is contacted at an aperture positionedat a side edge of the sensor for introducing the sample fluid into thesample chamber of the sensor. Moreover, the sensors may have any desiredelectrode configuration. For example, the sensors may have the workingand counter electrode on separate substrates and in a facingconfiguration. Alternatively, the sensors may have the working andcounter electrodes disposed on a single substrate in a coplanarconfiguration.

Referring to the Drawings in general and FIG. 3 and FIG. 4 inparticular, a first embodiment of a sensor 10 is schematicallyillustrated, herein shown in the shape of a strip. It is to beunderstood that the sensor may be any suitable shape. Sensor strip 10has a first substrate 12, a second substrate 14, and a spacer 15positioned therebetween. Sensor strip 10 includes at least one workingelectrode 24 and at least one counter electrode 22. Sensor strip 10 alsoincludes an optional insertion monitor 30.

Sensor strip 10 has a first, proximal end 10A and an opposite, distalend 10B. At proximal end 10A, sample to be analyzed is applied to sensor10. Proximal end 10A could be referred as ‘the fill end’, ‘samplereceiving end’, or similar. Distal end 10B of sensor 10 is configuredfor operable, and usually releasable, connecting to a device such as ameter.

Sensor strip 10 is a layered construction, in certain embodiments havinga generally rectangular shape, i.e., its length is longer than itswidth, although other shapes are possible as well, as noted above. Thelength of sensor strip 10 is from end 10A to end 10B.

The dimensions of a sensor may vary. In certain embodiments, the overalllength of sensor strip 10 may be no less than about 10 mm and no greaterthan about 50 mm. For example, the length may be between about 30 and 45mm; e.g., about 30 to 40 mm. It is understood, however that shorter andlonger sensor strips 10 could be made. In certain embodiments, theoverall width of sensor strip 10 may be no less than about 3 mm and nogreater than about 15 mm. For example, the width may be between about 4and 10 mm, about 5 to 8 mm, or about 5 to 6 mm. In one particularexample, sensor strip 10 has a length of about 32 mm and a width ofabout 6 mm. In another particular example, sensor strip 10 has a lengthof about 40 mm and a width of about 5 mm. In yet another particularexample, sensor strip 10 has a length of about 34 mm and a width ofabout 5 mm.

As provided above, sensor strip 10 has first and second substrates 12,14, non-conducting, inert substrates which form the overall shape andsize of sensor strip 10. Substrates 12, 14 may be substantially rigid orsubstantially flexible. In certain embodiments, substrates 12, 14 areflexible or deformable. Examples of suitable materials for substrates12, 14 include, but are not limited, to polyester, polyethylene,polycarbonate, polypropylene, nylon, and other “plastics” or polymers.In certain embodiments the substrate material is “Melinex” polyester.Other non-conducting materials may also be used.

Substrate 12 includes first or proximal end 12A and second or distal end12B, and substrate 14 includes first or proximal end 14A and second ordistal end 14B.

As indicated above, positioned between substrate 12 and substrate 14 maybe spacer 15 to separate first substrate 12 from second substrate 14. Insome embodiments, spacer 15 extends from end 10A to end 10B of sensorstrip 10, or extends short of one or both ends. Spacer 15 is an inertnon-conducting substrate, typically at least as flexible and deformable(or as rigid) as substrates 12, 14. In certain embodiments, spacer 15 isan adhesive layer or double-sided adhesive tape or film that iscontinuous and contiguous. Any adhesive selected for spacer 15 should beselected to not diffuse or release material which may interfere withaccurate analyte measurement. In certain embodiments, the thickness ofspacer 15 may be constant throughout, and may be at least about 0.01 mm(10 μm) and no greater than about 1 mm or about 0.5 mm. For example, thethickness may be between about 0.02 mm (20 μm) and about 0.2 mm (200μm). In one certain embodiment, the thickness is about 0.05 mm (50 μm),and about 0.1 mm (100 μm) in another embodiment.

The sensor includes a sample chamber for receiving a volume of sample tobe analyzed; in the embodiment illustrated, particularly in FIG. 3,sensor strip 10 includes sample chamber 20 having an inlet 21 for accessto sample chamber 20. In the embodiment illustrated, sensor strip 10 isa side-fill sensor strip, having inlet 21 present on a side edge ofstrip 10. Tip-fill sensors, having an inlet at, for example, end 10A,are also within the scope of this disclosure, as well as corner and topfilling sensors.

Sample chamber 20 is configured so that when a sample is provided inchamber 20, the sample is in electrolytic contact with both a workingelectrode and a counter electrode, which allows electrical current toflow between the electrodes to effect the electrolysis (electrooxidationor electroreduction) of the analyte.

Sample chamber 20 is defined by substrate 12, substrate 14 and spacer15; in many embodiments, sample chamber 20 exists between substrate 12and substrate 14 where spacer 15 is not present. Typically, a portion ofspacer 15 is removed to provide a volume between substrates 12, 14without spacer 15; this volume of removed spacer is sample chamber 20.For embodiments that include spacer 15 between substrates 12, 14, thethickness of sample chamber 20 is generally the thickness of spacer 15.

Sample chamber 20 has a volume sufficient to receive a sample ofbiological fluid therein. In some embodiments, such as when sensor strip10 is a small volume sensor, sample chamber 20 has a volume that istypically no more than about 1 μL, for example no more than about 0.5μL, and also for example, no more than about 0.25 μL. A volume of nomore than about 0.1 μL is also suitable for sample chamber 20, as arevolumes of no more than about 0.05 μL and about 0.03 μL.

As provided above, the thickness of sample chamber 20 correspondstypically to the thickness of spacer 15. Particularly for facingelectrode configurations, as in the sensor illustrated in FIG. 4, thisthickness is small to promote rapid electrolysis of the analyte, as moreof the sample will be in contact with the electrode surface for a givensample volume. In addition, a thin sample chamber 20 helps to reduceerrors from diffusion of analyte into the sample chamber during theanalyte assay, because diffusion time is long relative to themeasurement time, which may be about 5 seconds or less.

As provided above, the sensor includes a working electrode and at leastone counter electrode. The counter electrode may be a counter/referenceelectrode. If multiple counter electrodes are present, one of thecounter electrodes will be a counter electrode and one or more may bereference electrodes.

The sensor includes at least one working electrode positioned within thesample chamber. In FIG. 4, working electrode 24 is illustrated onsubstrate 14. In alternate embodiments, a working electrode is presenton a different surface or substrate, such as substrate 12. Workingelectrode 24 extends from the sample chamber 20, proximate first end10A, to the other end of the sensor 10, end 10B, as an electrodeextension called a “trace”. The trace provides a contact pad 25 forproviding electrical connection to a meter or other device to allow fordata and measurement collection, as will be described later. Contact pad25 may be positioned on a tab 27 that extends from the substrate onwhich working electrode 24 is positioned, such as substrate 12 or 14. Insome embodiments, a tab has more than one contact pad positionedthereon. In alternate embodiments, a single contact pad is used toprovide a connection to one or more electrodes; that is, multipleelectrodes are coupled together and are connected via one contact pad.

As provided above, at least a portion of working electrode 24 isprovided in sample chamber 20 for the analysis of analyte, inconjunction with the counter electrode.

Referring to FIG. 4 and in accordance with the present disclosure, a drycomposition including at least one analyte detection reagent (e.g., ananalyte-responsive enzyme and/or a redox mediator) and particulatematter (e.g., conductive particles, such as carbon nanopowder,conductive microspheres, and/or the like) may be disposed on a surfaceof an area of working electrode 24, e.g., all or a portion of thesurface of working electrode region 32. The dry composition may bedisposed on working electrode 24 prior to disposing spacer 15 onsubstrate 14. Alternatively, spacer 15 is first disposed on substrate 14(creating a channel having working electrode 24 as its base, similar tothe channel shown in FIGS. 2A-2E), followed by application of an aqueouscomposition that includes at least one detection reagent and particulatematter to all or a portion of the surface of working electrode region32. In accordance with the present disclosure, as the aqueouscomposition dries, the detection reagent(s) remain distributedsubstantially uniformly over the working electrode surface due to thepresence of the particulate matter in the composition (see, e.g., FIGS.2A-2E).

For sensor 10, at least one counter electrode is positioned on one offirst substrate 12 and second substrate 14 in the sample chamber. InFIG. 4, counter electrode 22 is illustrated on substrate 12. Counterelectrode 22 extends from the sample chamber 20, proximate end 10A, tothe other end of the sensor 10, end 10B, as an electrode extensioncalled a “trace”. The trace provides a contact pad 23 for providingelectrical connection to a meter or other device to allow for data andmeasurement collection, as will be described later. Contact pad 23 maybe positioned on a tab 26 that extends from the substrate on whichcounter electrode 22 is positioned, such as substrate 12. In someembodiments, a tab has more than one contact pad positioned thereon. Inalternate embodiments, a single contact pad is used to provide aconnection to one or more electrodes; that is, multiple electrodes arecoupled together and are connected via one contact pad.

Referring to FIG. 4 and in accordance with the present disclosure, apolymer layer may be disposed on a surface of an area of counterelectrode 22, e.g., all or a portion of the surface of counter electroderegion 34. Exemplary polymer materials and layers thereof are describedelsewhere herein. The polymer layer may serve as a physical solidbarrier to prevent conductive particles disposed on the surface of theworking electrode from forming a short connection between coplanar orfacing working and counter electrodes of the sensor. In certain aspects,the polymer layer, upon wetting by a fluid sample (e.g., a blood sampleof a subject), is able to conduct ions with a conductivity high enoughto not limit the current of the sensor, and also maintains its physicalintegrity when wetted by the fluid sample to prevent any shortingthroughout the entire analyte measurement period.

Working electrode 24 and counter electrode 22 may be disposed oppositeto and facing each other to form facing electrodes. See for example,FIG. 4, which has working electrode 24 on substrate 14 and counterelectrode 22 on substrate 12, forming facing electrodes. In thisconfiguration, the sample chamber is typically present between the twoelectrodes 22, 24. Working electrode 24 and counter electrode 22 mayalternately be positioned generally planar to one another, such as onthe same substrate, to form co-planar or planar electrodes.

In some instances, it is desirable to be able to determine when thesample chamber of the sensor is sufficiently filled with sample. Sensorstrip 10 may be indicated as filled, or substantially filled, byobserving a signal between an optional indicator electrode and one orboth of working electrode 24 or counter electrode 22 as sample chamber20 fills with fluid. When fluid reaches the indicator electrode, thesignal from that electrode will change. Suitable signals for observinginclude, for example, voltage, current, resistance, impedance, orcapacitance between the indicator electrode and, for example, workingelectrode 24. Alternatively, the sensor may be observed after filling todetermine if a value of the signal (e.g., voltage, current, resistance,impedance, or capacitance) has been reached indicating that the samplechamber is filled.

The optional indicator electrode may also be used to improve theprecision of the analyte measurements. The indicator electrode mayoperate as a working electrode or as a counter electrode orcounter/reference electrode. Measurements from the indicatorelectrode/working electrode may be combined (e.g., added or averaged)with those from the first counter/reference electrode/working electrodeto obtain more accurate measurements.

The sensor or equipment that the sensor connected is with (e.g., ameter) may include a signal (e.g., a visual sign or auditory tone) thatis activated in response to activation of the indicator electrode toalert the user that the sample chamber is beginning to fill with sampleand/or that the sample chamber is sufficiently filled with sample tomeasure the analyte concentration. The sensor or equipment may beconfigured to initiate a reading when the indicator electrode indicatesthat the sample chamber has been filled with or without alerting theuser. The reading may be initiated, for example, by applying a potentialbetween the working electrode and the counter electrode and beginning tomonitor the signals generated at the working electrode.

Referring to FIG. 5, an embodiment of an analyte sensor is illustratedas analyte sensor 10. The analyte sensor strip 10 has a first substrate12, a second substrate 14, and a spacer 15 positioned therebetween.Analyte sensor strip 10 includes at least one working electrode 22 andat least one counter electrode 24.

Analyte sensor strip 10 has a first, proximal end and an opposite,distal end. At proximal end, sample to be analyzed is applied to sensor10. Proximal end could be referred as “the fill end” or “samplereceiving end”. Distal end of sensor 10 is configured for operableconnection to a device such as a meter. Sensor strip 10 is a layeredconstruction, in certain embodiments having a generally rectangularshape, which is formed by first and second substrates 12, 14. Substrate12 includes first or proximal end 12A and second or distal end 12B, andsubstrate 14 includes first or proximal end 14A and second or distal end14B.

Sensor strip 10 includes sample chamber 20 having an inlet 21 for accessto sample chamber 20. Sensor strip 10 is a tip-fill sensor, having inlet21 at the proximal end. Sample chamber 20 is defined by substrate 12,substrate 14 and spacer 15. Generally opposite to inlet 21, throughsubstrate 12 is a vent 30 from sample chamber 20.

For sensor 10, at least one working electrode 22 is illustrated onsubstrate 14. Working electrode 22 extends from end 14A into samplechamber 20 to end 14B. Sensor 10 also includes at least one counterelectrode 24, in this embodiment on substrate 14. Counter electrode 24extends from sample chamber 20, proximate first proximal end to distalend. Working electrode 22 and counter electrode 24 are present on thesame substrate e.g., as planar or co-planar electrodes.

Referring to FIG. 5 and in accordance with the present disclosure, a drycomposition including at least one analyte detection reagent (e.g., ananalyte-responsive enzyme and/or a redox mediator) and particulatematter (e.g., conductive particles, such as carbon nanopowder,conductive microspheres, and/or the like) may be disposed on a surfaceof an area of working electrode 22, e.g., all or a portion of thesurface of working electrode area 36. The dry composition may bedisposed on working electrode 22 prior to disposing spacer 15 onsubstrate 14. Alternatively, spacer 15 is first disposed on substrate 14(creating a channel having working electrode 22 as a portion of itsbase, similar to the channel shown in FIGS. 2A-2E), followed byapplication of an aqueous composition that includes at least onedetection reagent and particulate matter to an exposed portion of thesurface of working electrode area 32. In accordance with the presentdisclosure, as the aqueous composition dries, the at least one detectionreagent remains distributed substantially uniformly over the workingelectrode surface to which the composition is applied, due to thepresence of the particulate matter in the composition (see, e.g., theprinciple schematically illustrated in FIGS. 2A-2E).

Also referring to FIG. 5 and in accordance with the present disclosure,a polymer layer may be disposed on a surface of an area of counterelectrode 24, e.g., all or a portion of the surface of counter electrodearea 38. Exemplary polymer materials and layers thereof are describedelsewhere herein. The polymer layer may serve as a physical solidbarrier to prevent conductive particles disposed on or in the vicinityof the surface of the working electrode from forming a short connectionbetween the working and counter electrodes of the sensor. In certainaspects, the polymer layer, upon wetting by a fluid sample (e.g., ablood sample of a subject), is able to conduct ions with a conductivityhigh enough to not limit the current of the sensor, and also maintainsits physical integrity when wetted by the fluid sample to prevent anyshorting throughout the entire analyte measurement period.

Sensors Having a Working Electrode with Increased Effective Surface Area

Embodiments of the present disclosure relate to sensors having a workingelectrode with increased effective surface area by disposition ofconductive particulate matter (e.g., conductive microspheres) on asurface of the working electrode of the sensor, such as in vitro or invivo analyte sensors. Also provided are methods of manufacturing theanalyte sensors and methods of using the analyte sensors in analytemonitoring.

The inventors of the present disclosure have found that disposing one ormore layers of conductive particles (e.g., conductive microspheres) on asurface of a working electrode increases the effective surface area ofthe working electrode. As a result of the increased effective surfacearea of the working electrode, higher peak currents, shorter test times,and/or more linear results may be obtained during operation of thesensor.

A working electrode surface having conductive microspheres disposedthereon is schematically illustrated in FIGS. 6A and 6B. FIG. 6A showsworking electrode surface 604 having a monolayer of conductivemicrospheres disposed thereon. The microspheres are in contact with theworking electrode surface, as well as with each other, such that eachsphere acts as an extension of the electrode surface. In other aspects,provided are sensors having a working electrode surface with more thanone layer of conductive microspheres disposed thereon. An example of aworking electrode surface having more than one layer of conductivemicrospheres is schematically illustrated in FIG. 6B, whereapproximately two layers (i.e., a bilayer) of conductive microspheresare disposed on the working electrode. In this aspect, conductivemicrospheres of a first layer makes contact with the working electrodesurface, with each other, and also with conductive microspheres of asecond layer. The conductive microspheres of the second layer makecontact with the conductive microspheres of the first layer, as well aseach other. Whether one, two or more layers of conductive microspheresare provided on the surface of the working electrode, the result is thatall or a majority of the spheres act as an extension of the workingelectrode surface, thereby increasing the effective surface area of theworking electrode.

According to certain embodiments, one or more layers of conductiveparticles are disposed on a surface of the working electrode (e.g., bydepositing a conductive particle-containing suspension on the workingelectrode surface, followed by drying), and an analyte detectionsolution that includes one or more analyte detection reagents (e.g., ananalyte-responsive enzyme and/or a redox mediator) is deposited over theconductive particles. In other aspects, one or more layers of conductiveparticles are disposed on the surface of the working electrode byapplying a conductive particle suspension that also includes one or moreanalyte detection reagents (e.g., an analyte-responsive enzyme and/or aredox mediator).

The desired number of layers of conductive particles on the workingelectrode surface may be achieved using any suitable approach. Forexample, when a particle-containing suspension is deposited on a workingelectrode surface, the particle density of the suspension may becontrolled to achieve a monolayer, bilayer, trilayer, or more layers ofconductive particles on the surface. Whether or not a particularparticle density achieves the desired number of layers may bedetermined, e.g., via microscopic imaging of the particles disposed onthe electrode surface. In certain aspects, the desired number of layersof conductive particles is achieved by first determining the particledensity of a particle suspension required to achieve a monolayer ofparticles on the electrode surface (for particles having a particulardiameter), and then increasing the particle density by a factor of two,three or more to achieve a bilayer, trilayer, or more layers,respectively, of conductive particles on the surface.

Other approaches for achieving the desired number of particle layers onthe working electrode surface include inkjet deposition and spraydeposition, both of which can be performed in multiple passes, such thatthe preceding pass is allowed to dry before a subsequent pass/layer isapplied.

The conductive particles may be deposited over the entire surface of theworking electrode. Alternatively, the surface of the working electrodeon which the conductive particles are deposited is substantially limitedto all or a portion of the working electrode surface positioned withinthe sample chamber of the sensor. For example, referring to the sensorembodiments shown in FIG. 4 and FIG. 5, the one or more layers ofconductive particles may be disposed on the working electrode regionsubstantially or entirely limited to working electrode regions 32 and36, respectively, or a sub-region thereof.

Sensors Having Reduced Interference from Sample Fluid Components

Embodiments of the present disclosure relate to sensors havingparticulate matter (e.g., carbon nanopowder, microspheres, and/or thelike) in a sample chamber of the sensor, such as in vitro or in vivoanalyte sensors, such that the particulate matter excludes componentsthat interfere with analyte measurement from entering the samplechamber. Also provided are methods of manufacturing the analyte sensorsand methods of using the analyte sensors in analyte monitoring.

According to one aspect, sensors are provided that include a sufficientamount of particulate matter in the sample chamber such that the samplechamber is substantially filled with the particulate matter and, as aresult, the entry into the sample chamber of interfering substances in asample fluid is decreased or prevented during operation of the sensor.With reference to FIG. 4 and FIG. 5, the particulate matter (e.g.,conductive microspheres) may be disposed in—and substantially fill—thesample chamber region formed by overlaying the spacer on the substratethat includes the working electrode, such that upon overlaying the topsubstrate onto the spacer, the particles substantially fill theresulting sample chamber. A number of variations are possible. Forexample, provided are sensors where a central or terminal portion of thesample chamber may be substantially devoid of particles, and themajority (or all) of the particles are disposed at the portion of thesample chamber proximal to the sample entry port/aperture.

The volume of the sample chamber occupied by the particulate matter(e.g., conductive particles, such as conductive microspheres) may bedetermined during the sensor manufacturing process, e.g., by depositinga suspension of particulate matter (optionally with detection reagents)in which the concentration of the particulate matter in the suspensiondetermines the volume of the sample chamber occupied by the particulatematter. The particulate matter may, for example, occupy from about 10%to about 90% of the volume of the sample chamber, e.g., from about 25%to about 90%, from about 50% to about 90%, from about 75% to about 90%,or from about 80% to about 90% of the volume of the sample chamber. Incertain aspects, the volume of the sample chamber occupied by theparticulate matter is greater than about 90%, e.g., greater than about95%.

Any particulate matter suitable for decreasing or preventing entry of anundesired interfering substance may be used. For example, based on thesize of the interfering substance, particles having a diameter within aparticular range may be chosen such that—when the particles are disposedin the sample chamber—the spaces between the particles are sufficientlysmall such that entry of the interfering substance into the samplechamber is prevented. According to one embodiment, the sample fluid isblood and red blood cells (or “erythrocytes”) constitute the undesiredinterfering substance. The dimensions of red blood cells of variouspopulation of interest (e.g., humans) are known in the art. For example,the diameter of human red blood cells ranges from 6-8 μm. Accordingly,when it is desired to decrease or prevent entry of human red blood cellsinto the sample chamber, the diameter of the particles may be chosen sothat the maximum dimension of the spaces (or “voids”) between theparticles is less than or equal to about 6-8 μm. Generally, the particlesize should be chosen such that entry of the undesired interferingsubstance into the sample chamber is sufficiently decreased orprevented, but that the particles are not so small that the flow rate ofa fluid sample into the sample chamber is markedly reduced as a resultof the particles in the sample chamber excessively impeding the flow ofthe sample fluid. As such, the particles may be chosen according to therequirements and limitations of the particular sensor (and analytemonitoring system) to be used.

Exemplary features and components of the sensors provided by the presentdisclosure are described in greater detail below. It will be appreciatedthat any such features and components may be employed—or present in—anyof the sensors described above, including sensors having improveduniformity of distribution of one or more analyte detection reagents,increased effective working electrode surface area, and/or reduced entryof interfering components into the sample chamber.

Working Electrode

As summarized previously herein, an analyte sensor includes a workingelectrode and a reference/counter electrode, comprising a first portionlocated in the sample chamber and a second portion for connection to ameter. The working electrode may be formed from a suitable conductingmaterial. The conducting material may have relatively low electricalresistance and may be electrochemically inert over the potential rangeof the sensor during operation and substantially transparent. In certainaspects, the working electrode includes a material selected from thegroup consisting of gold, carbon, platinum, ruthenium, palladium,silver, silver chloride, silver bromide, and combinations thereof. Theworking electrode may be a thin layer of gold, tin oxide, platinum,ruthenium dioxide or palladium, indium tin oxide, zinc oxide, fluorinedoped tin oxide, as well as other non-corroding materials known to thoseskilled in the art. The working electrode can be a combination of two ormore conductive materials. For example, the working electrode may beconstructed from thin layer of gold in the sample chamber and of carbonoutside the sample chamber.

The working electrode can be applied on a substrate by any of a varietyof methods, including by being deposited, such as by vapor deposition orvacuum deposition or otherwise sputtered, printed on a flat surface orin an embossed or otherwise recessed surface, transferred from aseparate carrier or liner, etched, or molded. Suitable methods ofprinting include screen-printing, piezoelectric printing, ink jetprinting, laser printing, photolithography, painting, gravure rollprinting, transfer printing, and other known printing methods.

Particles

Any particulate matter suitable for improving the distribution ofanalyte detection reagents, increasing the effective surface area of aworking electrode, and/or preventing interfering substances fromentering a sample chamber, may be used in the sensors of the presentdisclosure.

In certain embodiments, the particles comprise nanomaterials (e.g.,conductive nanomaterials). Exemplary nanomaterials include, but are notlimited to, aluminum nanomaterial, carbon nanomaterial, cobalt carboncoated nanomaterial, copper nanomaterial, copper nanomaterial,copper-zinc alloy nanomaterial, diamond nanomaterial, gold nanomaterial,iron nanomaterial, iron-nickel alloy nanomaterial, molybdenumnanomaterial, magnesium nanomaterial, nickel nanomaterial, palladiumnanomaterial, platinum nanomaterial, silver nanomaterial, silver-copperalloy nanomaterial, tantalum nanomaterial, tin nanomaterial, indiumdoped tin oxide nanomaterial, titanium nanomaterial, titanium nitridenanomaterial, tungsten nanomaterial, zinc nanomaterial, calcium oxidenanomaterial, hydroxyapatite nanomaterial, indium nanomaterial, silicananomaterial, silicon nanomaterial, silicon dioxide nanomaterial,silicon nitride nanomaterial, silicon carbide nanomaterial, and thelike. Exemplary nanomaterials may also include polymers such aspolyethylene, polymethylene, polypropylene, or polystyrene, wherein thepolymer is not covalently conjugated to components of the analytesensor. Other exemplary nanomaterials are well known and commerciallyavailable from suppliers, such as, for example, Sigma-Aldrich.

When the particles comprise a nanomaterial, the nanomaterial may be ananopowder or a nanoparticle, such as a carbon nanopowder. In suchembodiments, the nanopowder or nanoparticle will have particles having adiameter of from about 1 nm to about 300 nm, including about 10 nm toabout 290 nm, about 15 nm to about 275 nm, about 20 nm to about 250 nm,about 30 nm to about 225 nm, about 35 nm to about 200 nm, about 40 nm toabout 175 nm, about 45 nm to about 150 nm, about 50 nm to about 125 nm,about 55 nm to about 100 nm, and about 60 nm to about 75 nm.

In certain aspects, the particles are nanospheres or microspheres. Incertain aspects, the particles are conductive microspheres. Theconductive microspheres may comprise a polymer core made of a polymermaterial selected from, but not limited to, polyethylene, polymethylene,polypropylene, polystyrene, and combinations thereof. The polymer coremay be coated with one or more conductive layers. The conductive layermay include any suitable conducting material, including but not limitedto, gold, carbon, platinum, ruthenium, palladium, silver, silverchloride, silver bromide, and combinations thereof.

According to certain embodiments, the microspheres are hollow or porous.The pores of porous microspheres, for example, provide additionalsurface area for disposing a conductive coating and/or one or moreanalyte detection reagents (e.g., an analyte-responsive enzyme and/orredox mediator) onto the surface of the microsphere. This additionalsurface area may improve the performance of the sensor by enhancingconductivity, providing greater access of the analyte to the detectionreagents, and the like.

When the sensors include microspheres (e.g., conductive microspheres),the average diameter of the conductive microspheres may vary. Forexample, the conductive microspheres may have a diameter of from about0.5 μm to about 100 μm, including about 1 μm to about 50 μm, about 2.5μm to about 25 μm, and about 5 μm to about 10 μm. In certain aspects,sensors of the present disclosure include conductive microspheres havingan average diameter of about 5 μm.

Counter Electrode

The counter electrode may be constructed in a manner similar to theworking electrode. As used herein, the term “counter electrode” refersto an electrode that functions as a counter electrode, or both areference electrode and a counter electrode. The counter electrode canbe formed, for example, by depositing electrode material onto asubstrate. The material of the counter electrode may be deposited by avariety of methods such as those described above for the workingelectrode. In certain aspects, the counter electrode includes a materialselected from the group consisting of gold, carbon, platinum, ruthenium,palladium, silver, silver chloride, silver bromide, and combinationsthereof. For example, suitable materials for the counter electrodeinclude Ag/AgCl or Ag/AgBr printed on a non-conducting substrate.According to certain embodiments, the counter electrode comprises a thinconductive layer such as gold, tin oxide, indium tin oxide, layered withAgCl or AgBr, for example.

Polymer Layer

As described elsewhere herein, sensors of the present disclosure mayinclude conductive particles in the sample chamber to improve thedistribution of one or more detection reagents, increase the effectivesurface area of the working electrode, and/or inhibit entry of certaininterfering substances into the sample chamber. However, if theconductive particles are misplaced outside the sample chamber region, ifthe particles are excessively stacked in the sample chamber region, orgenerally if the particles form a physical and/or electrical connectionbetween the working and counter electrode, the particles can cause anelectrical short between the working electrode and counter electrodeduring operation of the sensor, preventing the sensor from functioningproperly.

The inventors of the present disclosure have discovered that disposing apolymer layer onto the counter electrode of a sensor having conductivemicroparticles reduces or eliminates electrical shorting between theworking and counter electrodes. The polymer layer (or coating) serves asa physical solid barrier to prevent the loose conducting particles fromforming a short connection between coplanar or facing working andcounter electrodes of the sensor.

In certain aspects, the polymer layer, upon wetting by a fluid sample(e.g., a blood sample of a subject), is able to conduct ions with aconductivity high enough to not limit the current of the sensor, andalso maintains its physical integrity when wetted by the fluid sample toprevent any shorting throughout the entire analyte measurement period.One approach to improve the physical integrity of the polymer is tocross-link the polymer using compatible cross-linkers.

According to one embodiment, the polymer layer is a copolymer, such as acopolymer of vinyl pyridine and styrene grafted with propyl sulfonateand polyethylene glycol (PEG) having the following formula:

In the above polymer, the presence of the ionic moieties and PEG confersgood ionic conductivity upon swelling with water. In addition, thispolymer can be readily cross-linked with multi-epoxy functionalizedcross-linkers to enhance its physical integrity when exposed to a fluidsample.

Any suitable polymer/copolymer, e.g., a polymer or copolymer havingsufficient ion conductivity and/or adequate physical integrity under wetconditions, may be used. For example, the counter electrode may becoated with a polymer formed using a resin, such as an aliphaticurethane polymer, which is a polyurethane-based resin containingcarboxyl functional groups.

Additional polymers that find use in the sensors described hereininclude, but are not limited to, those described in U.S. Pat. No.6,932,894 and U.S. Patent Application Publication No. 2008/0179187, thedisclosures of which are incorporated herein in their entireties for allpurposes.

Electrode Configuration

A variety of analyte sensor electrode configurations are known in theart which may be suitable for use in the disclosed analyte sensors. Forexample, suitable configurations can include configurations having aworking electrode positioned in opposition to a reference/counterelectrode or configurations having the working electrode positionedcoplanar with the reference/counter electrode. Additional suitableelectrode configurations include, but are not limited to, thosedescribed in U.S. patent application Ser. No. 11/461,725, filed Aug. 1,2006; U.S. patent application Ser. No. 12/102,374, filed Apr. 14, 2008;U.S. Patent Application Publication No. 2007/0095661; U.S. PatentApplication Publication No. 2006/0091006; U.S. Patent ApplicationPublication No. 2006/0025662; U.S. Patent Application Publication No.2008/0267823; U.S. Patent Application Publication No. 2007/0108048; U.S.Patent Application Publication No. 2008/0102441; U.S. Patent ApplicationPublication No. 2008/0066305; U.S. Patent Application Publication No.2007/0199818; U.S. Patent Application Publication No. 2008/0148873; U.S.Patent Application Publication No. 2007/0068807; U.S. Patent ApplicationPublication No. 2009/0095625; U.S. Pat. Nos. 6,616,819; 6,143,164; and6,592,745; the disclosures of each of which are incorporated herein byreference in their entireties for all purposes.

Analytes

A variety of analytes can be detected and quantified using the analytesensors disclosed herein including, but not limited to, glucose, blood(3-ketone, ketone bodies, lactate, acetyl choline, amylase, bilirubin,cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK-MB),creatine, DNA, fructosamine, glutamine, growth hormones, hormones,ketones, lactate, peroxide, prostate-specific antigen, prothrombin, RNA,thyroid stimulating hormone, and troponin, in sample of body fluid.Analyte sensors may also be configured to detect and/or quantify drugs,such as, for example, antibiotics (e.g., gentamicin, vancomycin, and thelike), digitoxin, digoxin, drugs of abuse, theophylline, and warfarin.Assays suitable for determining the concentration of DNA and/or RNA aredisclosed in U.S. Pat. Nos. 6,281,006 and 6,638,716, the disclosures ofeach of which are incorporated by reference herein.

Analyte-Responsive Enzyme

The disclosed analyte sensors may include in the sample chamber ananalyte-responsive enzyme which is capable of transferring electrons toor from a redox mediator and the analyte. For example, a glucose oxidase(GOD) or glucose dehydrogenase (GDH) can be used when the analyte isglucose. A lactate oxidase can be used when the analyte is lactate.These enzymes catalyze the electrolysis of an analyte by transferringelectrons between the analyte and the electrode via the redox mediator.In one embodiment, the analyte-responsive enzyme is disposed on theworking electrode. In certain embodiments, the analyte-responsive enzymeis immobilized on the working electrode. This is accomplished, forexample, by cross linking the analyte-responsive enzyme with a redoxmediator on the working electrode, thereby providing a sensing layer onthe working electrode. In an alternative embodiment, theanalyte-responsive enzyme is disposed adjacent to the electrode.Generally, the analyte-responsive enzyme and redox mediator arepositioned in close proximity to the working electrode in order toprovide for electrochemical communication between the analyte-responsiveenzyme and redox mediator and the working electrode. Generally, theanalyte-responsive enzyme and redox mediator are positioned relative tothe reference/counter electrode such that electrochemical communicationbetween the analyte-responsive enzyme and the redox mediator and thereference/counter electrode is minimized. Additional analyte-responsiveenzymes and cofactors which may be used in connection with the disclosedanalyte sensors are described in U.S. Pat. No. 6,736,957, the disclosureof which is incorporated by reference herein.

In some embodiments, in order to facilitate the electrochemical reactionof the analyte sensor the sample chamber also includes an enzymeco-factor. For example, where the analyte-responsive enzyme is glucosedehydrogenase (GDH), suitable cofactors include pyrroloquinoline quinone(PQQ), nicotinamide adenine dinucleotide NAD+ and flavin adeninedinucleotide (FAD).

In certain embodiments, the analyte detected and/or measured by thesensor described herein may be ketone and the enzyme included in thesensor is hydroxybutyrate dehydrogenase.

Redox Mediator

In addition to the analyte-responsive enzyme, the sample chamber mayinclude a redox mediator. In one embodiment, the redox mediator isimmobilized on the working electrode. Materials and methods forimmobilizing a redox mediator on an electrode are provided in U.S. Pat.No. 6,592,745, the disclosure of which is incorporated by referenceherein. In an alternative embodiment, the redox mediator is disposedadjacent to the working electrode.

The redox mediator mediates a current between the working electrode andthe analyte when present. The mediator functions as an electron transferagent between the electrode and the analyte.

Almost any organic or organometallic redox species can be used as aredox mediator. In general, suitable redox mediators are rapidlyreducible and oxidizable molecules having redox potentials a few hundredmillivolts above or below that of the standard calomel electrode (SCE),and typically not more reducing than about −200 mV and not moreoxidizing than about +400 mV versus SCE. Examples of organic redoxspecies are quinones and quinhydrones and species that in their oxidizedstate have quinoid structures, such as Nile blue and indophenol.Unfortunately, some quinones and partially oxidized quinhydrones reactwith functional groups of proteins such as the thiol groups of cysteine,the amine groups of lysine and arginine, and the phenolic groups oftyrosine which may render those redox species unsuitable for some of thesensors of the present invention, e.g., sensors that will be used tomeasure analyte in biological fluids such as blood.

In certain cases, mediators suitable for use in the analyte sensors havestructures which prevent or substantially reduce the diffusional loss ofredox species during the period of time that the sample is beinganalyzed. Suitable redox mediators include a redox species bound to apolymer which can in turn be immobilized on the working electrode.Useful redox mediators and methods for producing them are described inU.S. Pat. Nos. 5,262,035; 5,264,104; 5,320,725; 5,356,786; 6,592,745;and 7,501,053, the disclosure of each of which is incorporated byreference herein. Any organic or organometallic redox species can bebound to a polymer and used as a redox mediator. In certain cases, theredox species is a transition metal compound or complex. The transitionmetal compounds or complexes may be osmium, ruthenium, iron, and cobaltcompounds or complexes. In certain cases, the redox mediator may be anosmium compounds and complex.

One type of non-releasable polymeric redox mediator contains a redoxspecies covalently bound in a polymeric composition. An example of thistype of mediator is poly(vinylferrocene).

Alternatively, a suitable non-releasable redox mediator contains anionically-bound redox species. Typically, these mediators include acharged polymer coupled to an oppositely charged redox species. Examplesof this type of mediator include a negatively charged polymer such asNafion® (Dupont) coupled to a positively charged redox species such asan osmium or ruthenium polypyridyl cation. Another example of anionically-bound mediator is a positively charged polymer such asquaternized poly(4-vinyl pyridine) or poly(l-vinyl imidazole) coupled toa negatively charged redox species such as ferricyanide or ferrocyanide.

In another embodiment, the suitable non-releasable redox mediatorsinclude a redox species coordinatively bound to the polymer. Forexample, the mediator may be formed by coordination of an osmium orcobalt 2,2′-bipyridyl complex to poly(l-vinyl imidazole) or poly(4-vinylpyridine).

The redox mediator may be a osmium transition metal complex with one ormore ligands having a nitrogen-containing heterocycle such as2,2′-bipyridine, 1,10-phenanthroline or derivatives thereof.Furthermore, the redox mediator may also have one or more polymericligands having at least one nitrogen-containing heterocycle, such aspyridine, imidazole, or derivatives thereof. These mediators exchangeelectrons rapidly between each other and the electrodes so that thecomplex may be rapidly oxidized and reduced.

In particular, it has been determined that osmium cations complexed withtwo ligands containing 2,2′-bipyridine, 1,10-phenanthroline, orderivatives thereof, the two ligands not necessarily being the same, andfurther complexed with a polymer having pyridine or imidazole functionalgroups form particularly useful redox mediators in the small volumesensors. Derivatives of 2,2′-bipyridine for complexation with the osmiumcation may be 4,4′-dimethyl-2,2′-bipyridine and mono-, di-, andpolyalkoxy-2,2′-bipyridines, such as 4,4′-dimethoxy-2,2′-bipyridine,where the carbon to oxygen ratio of the alkoxy groups is sufficient toretain solubility of the transition metal complex in water. Preferredderivatives of 1,10-phenanthroline for complexation with the osmiumcation are 4,7-dimethyl-1,10-phenanthroline and mono-, di-, andpolyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline, where the carbon to oxygen ratio ofthe alkoxy groups is sufficient to retain solubility of the transitionmetal complex in water. Exemplary polymers for complexation with theosmium cation include poly(l-vinyl imidazole), e.g., PVI, andpoly(4-vinyl pyridine), e.g., PVP, either alone or with a copolymer.Most preferred are redox mediators with osmium complexed withpoly(l-vinyl imidazole) alone or with a copolymer.

Suitable redox mediators have a redox potential between about −150 mV toabout +400 mV versus the standard calomel electrode (SCE). For example,the potential of the redox mediator can be between about −100 mV and+100 mV, e.g., between about −50 mV and +50 mV. In one embodiment,suitable redox mediators have osmium redox centers and a redox potentialmore negative than +100 mV versus SCE, e.g., the redox potential is morenegative than +50 mV versus SCE, e.g., is near −50 mV versus SCE.

In one embodiment, the redox mediators of the disclosed analyte sensorsare air-oxidizable. This means that the redox mediator is oxidized byair, e.g., so that at least 90% of the mediator is in an oxidized stateprior to introduction of sample into the sensor. Air-oxidizable redoxmediators include osmium cations complexed with two mono-, di-, orpolyalkoxy-2,2′-bipyridine or mono-, di-, orpolyalkoxy-1,10-phenanthroline ligands, the two ligands not necessarilybeing the same, and further complexed with polymers having pyridine andimidazole functional groups. In particular,Os[4,4′-dimethoxy-2,2′-bipyridine]₂Cl^(+/+2) complexed with poly(4-vinylpyridine) or poly(l-vinyl imidazole) attains approximately 90% or moreoxidation in air.

In one specific embodiment, the redox mediator is 1,10Phenanthrolene-5,6-dione (PQ).

To prevent electrochemical reactions from occurring on portions of theworking electrode not coated by the mediator, a dielectric may bedeposited on the electrode surrounding the region with the bound redoxmediator. Suitable dielectric materials include waxes and non-conductingorganic polymers such as polyethylene. Dielectric may also cover aportion of the redox mediator on the electrode. The covered portion ofthe mediator will not contact the sample, and, therefore, will not be apart of the electrode's working surface.

Although it can be advantageous to minimize the amount of redox mediatorused, the range for the acceptable amount of redox mediator typicallyhas a lower limit. The minimum amount of redox mediator that may be usedis the concentration of redox mediator that is necessary to accomplishthe assay within a desirable measurement time period, for example, nomore than about 5 minutes, or no more than about 1 minute, or no morethan about 30 seconds, or no more than about 10 seconds, or no more thanabout 5 seconds, or no more than about 3 seconds, or no more than about1 second or less.

The analyte sensor can be configured (e.g., by selection of redoxmediator, positioning of electrodes, etc.) such that the sensor signalis generated at the working electrode with a measurement period of nogreater than about 5 minutes and such that a background signal that isgenerated by the redox mediator is no more than five times a signalgenerated by oxidation or reduction of 5 mM analyte. In someembodiments, the analyte sensor is configured such that the backgroundsignal that is generated by the redox mediator is less than the signalgenerated by oxidation or reduction of 5 mM glucose. In someembodiments, the background that is generated by the redox mediator isno more than 25% of the signal generated by oxidation or reduction of 5mM analyte, e.g., no more than 20%, no more than 15% or no more than 5%.In certain embodiments, the analyte is glucose and the background thatis generated by the redox mediator is no more than 25% of the signalgenerated by oxidation or reduction of 5 mM glucose, e.g., no more than20%, no more than 15% or no more than 5% of the signal generated byelectrolysis of glucose.

Sorbent Material

The sample chamber may be empty prior to entry of the sample.Optionally, the sample chamber can include a sorbent material to sorband hold a fluid sample during detection and/or analysis. Suitablesorbent materials include polyester, nylon, cellulose, and cellulosederivatives such as nitrocellulose. The sorbent material facilitates theuptake of small volume samples by a wicking action which may complementor replace any capillary action of the sample chamber. In addition oralternatively, a portion or the entirety of the wall of the samplechamber may be covered by a surfactant, such as, for example, Zonyl FSO.

In some embodiments, the sorbent material is deposited using a liquid orslurry in which the sorbent material is dissolved or dispersed. Thesolvent or dispersant in the liquid or slurry may then be driven off byheating or evaporation processes. Suitable sorbent materials include,for example, cellulose or nylon powders dissolved or dispersed in asuitable solvent or dispersant, such as water. The particular solvent ordispersant should also be compatible with the material of the electrodes(e.g., the solvent or dispersant should not dissolve the electrodes).

One of the functions of the sorbent material is to reduce the volume offluid needed to fill the sample chamber of the analyte sensor. Theactual volume of sample within the sample chamber is partiallydetermined by the amount of void space within the sorbent material.Typically, suitable sorbents consist of about 5% to about 50% voidspace. In one embodiment, the sorbent material consists of about 10% toabout 25% void space.

Fill Assist

The analyte sensors can be configured for top-filling, tip-filling,corner-filling, and/or side-filling. In some embodiments, the analytesensors include one or more optional fill assist structures, e.g., oneor more notches, cut-outs, indentations, and/or protrusions, whichfacilitate the collection of the fluid sample. For example, the analytesensor can be configured such that the proximal end of the analytesensor is narrower than the distal end of the analyte sensor. In onesuch embodiment, the analyte sensor includes a tapered tip at theproximal end of the analyte sensor, e.g., the end of the analyte sensorthat is opposite from the end that engages with a meter.

Additional fill assist structures are described in U.S. PatentPublication No. 2008/0267823, the disclosure of which is incorporated byreference herein; and U.S. patent application Ser. No. 11/461,725, filedAug. 1, 2006, the disclosure of which is incorporated by referenceherein.

Signal Enhancement

In certain cases, the analyte sensor comprising particulate matter(e.g., conductive particles) on a working electrode surface provides asignal (e.g., a peak current) from electrolysis of an analyte in asample that is at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 120%, or 130% higherthan the signal generated from electrolysis of the analyte in the sampleusing a similar analyte sensor but not comprising particulate matter(e.g., conductive particles) on a working electrode surface. In certainembodiments, the higher signal is generated within 0.01 second, or 0.03second, or 0.01 second, or 0.3 second, or 0.6 second, or 1 second, or1.3 seconds, or 1.6 seconds, or 2 seconds, or 3 seconds, or 4 seconds,or 5 seconds, or more of applying the sample to the analyte sensor.

As used herein, “signal” refers to current, charge, resistance, voltage,impedance, or log or integrated values thereof that is related to theconcentration of the analyte being analyzed by the sensor.

Accuracy

In certain embodiments of the present disclosure, inclusion ofparticulate matter (e.g., conductive particles) on a working electrodesurface results in an increase in the accuracy of the analytemeasurements from the sensor. For example, inclusion of particulatematter (e.g., conductive particles) on a working electrode surface mayresult in better correlation between the analyte concentration asdetermined by an in vitro analyte monitoring device (e.g., based onsignals detected from the in vitro analyte sensor by a device operablyconnected to the sensor, for example, a meter) and a reference analyteconcentration. In certain instances, inclusion of particulate matter(e.g., conductive particles) on a working electrode surface results inanalyte concentrations as determined by the signals detected from theanalyte sensor that are within 50% of a reference value, such as within40% of the reference value, including within 30% of the reference value,or within 20% of the reference value, or within 10% of the referencevalue, or within 5% of the reference value, or within 2% of thereference value, or within 1% of the reference value. In some cases, 75%of the analyte sensors as described herein demonstrate the accuracy(e.g., is within a percentage of a reference value, as described above).In some cases, 80% or more, or 90% or more, including 95% or more, or97% or more, or 99% or more of the analyte sensors as described hereindemonstrate the accuracy (e.g., is within a percentage of a referencevalue, as described above).

As an alternative measure of accuracy, in some cases, inclusion ofparticulate matter (e.g., conductive particles) on a working electrodesurface results in analyte concentrations as determined by the signalsdetected from the analyte sensor that are within Zone A of the ClarkeError Grid Analysis. For example, inclusion of particulate matter (e.g.,conductive particles) on a working electrode surface may result inanalyte concentrations as determined by the signals detected from theanalyte sensor that are within Zone A of the Clarke Error Grid Analysisfor 75% or more of the analyte sensors, such as 80% or more, or 90% ormore, including 95% or more, or 97% or more, or 99% or more of theanalyte sensors. In certain instances, inclusion of conductive particlesin the sample chamber results in analyte concentrations as determined bythe signals detected from the analyte sensor that are within Zone A orZone B of the Clarke Error Grid Analysis. For example, inclusion ofconductive particles in the sample chamber may result in analyteconcentrations as determined by the signals detected from the analytesensor that are within Zone A or Zone B of the Clarke Error GridAnalysis for 75% or more of the analyte sensors, such as 80% or more, or90% or more, including 95% or more, or 97% or more, or 99% or more ofthe analyte sensors. Further information regarding the Clarke Error GridAnalysis is found in Clarke, W. L. et al. “Evaluating Clinical Accuracyof Systems for Self-Monitoring of Blood Glucose” Diabetes Care, vol. 10,no. 5, 1987: 622-628.

Methods of Determining Analyte Concentration

The sensors described herein find use in methods for determining theconcentration of an analyte in a fluid sample from a subject. Generally,these methods include contacting a fluid sample with the sensor,generating a sensor signal at the working electrode, and determining theconcentration of the analyte using the sensor signal. It will beunderstood that the subject methods may employ any of the sensorsdescribed herein, e.g., sensors having improved uniformity ofdistribution of one or more analyte detection reagents, increasedeffective working electrode surface area, and/or reduced entry ofinterfering components into the sample chamber.

A variety of approaches may be employed to determine the concentrationof the analyte. In certain aspects, an electrochemical analyteconcentration determining approach is used. For example, determining theconcentration of the analyte using the sensor signal may be performed bycoulometric, amperometric, potentiometric, or any other convenientelectrochemical detection technique.

According to certain embodiments, the subject methods include obtainingthe sample from a subject. When the sample is a blood sample, the samplemay be obtained, e.g., using a lancet to create an opening in a skinsurface at which blood subsequently presents. The blood sample may beobtained from the finger of a subject. Alternatively, the blood samplemay be obtained from a region of the subject having a lower nerve enddensity as compared to a finger. Obtaining a blood sample from a regionhaving a lower nerve end density as compared to a finger is generally aless painful approach for obtaining a blood sample and may improvepatient compliance, e.g., in the case of a diabetes patient whereregular monitoring of blood glucose levels is critical for diseasemanagement.

Methods of Making Analyte Sensors

Also provided by the present disclosure are methods of manufacturinganalyte sensors. In certain aspects, methods are provided that includeforming a working electrode on a first substrate, forming a spacer layeron the first substrate, the spacer layer defining a sample chamberregion on the first substrate, and applying a reagent composition on asurface of the working electrode in the sample chamber region. Thereagent composition may include one or more analyte detection reagents(e.g., an analyte-responsive enzyme and/or a redox mediator) andparticulate matter (e.g., conductive microspheres), where theparticulate matter provides for even distribution of the detectionreagent(s) as the reagent composition dries on the working electrode.

The sample chamber region on the first substrate includes at least aportion of a working electrode surface. Generally, the reagentcomposition is applied to all or a portion of the working electrodesurface in the sample chamber region, thereby generating a modifiedworking electrode surface in the sample chamber region on which one ormore analyte detection reagents are substantially uniformly distributed.

Optionally, the methods further comprise disposing a counter electrodeon the first substrate (e.g., on region of the first substrate distinctfrom the region on which the working electrode is disposed), oralternatively, disposing a counter electrode on a second substrate to beoverlayed on the first substrate (thereby generating a facing electrodepair). Manufacturing the sensor is generally completed by overlaying asecond substrate on the spacer layer and singulating individual sensors(e.g., by dye cutting, etc.) from the starting substrate material.General approaches for manufacturing analyte sensors are known in theart and are described, e.g., in U.S. Pat. No. 7,866,026, the fulldisclosure of which is incorporated herein by reference in its entiretyfor all purposes.

The present disclosure also provides methods of manufacturing analytesensors, which methods include forming a working electrode on a firstsubstrate, and disposing one or more layers of conductive material(e.g., conductive microspheres) on the working electrode, where the oneor more layers make up an ordered array of conductive microspheres. Byusing conductive material (e.g., conductive microspheres) having aconsistent size and shape, the conductive material is capable of beingstacked into ordered arrays, with easily tailored surface area and voidvolume. For example, the number of layers of conductive microspheres maybe selected to provide a desired effective surface area of the workingelectrode (e.g., where the effective surface area can be increased ordecreased by increasing or decreasing the number of layers of conductivemicrospheres, respectively). Alternatively, or additionally, the sizedistribution of the conductive microspheres may be selected to provide adesired void volume within the array of conductive microspheres.

Also provided are methods of manufacturing analyte sensors, whichmethods include forming a working electrode on a first substrate, anddisposing one or more layers of conductive microspheres on the workingelectrode, where a number of layers of the one or more layers ofconductive microspheres is selected to provide a desired effectivesurface area of the working electrode. Optionally, the one or morelayers make up an ordered array of conductive microspheres. The sizedistribution of the conductive microspheres may be selected to provide adesired void volume within the one or more layers of conductivemicrospheres.

In addition, the present disclosure provides of manufacturing analytesensors, which methods include forming a working electrode on a firstsubstrate, and disposing one or more layers of conductive microsphereson the working electrode, where a size distribution of the conductivemicrospheres is selected to provide a desired void volume within the oneor more layers of conductive microspheres. The number of layers of theone or more layers of conductive microspheres may be selected to providea desired effective surface area of the working electrode. The one ormore layers optionally make up an ordered array of conductivemicrospheres.

Utility

The subject sensors and methods find use in a variety of differentapplications where, e.g., the accurate determination of an analyteconcentration by an analyte sensor is desired. For example, the methodsare useful for obtaining and accurately determining the concentration ofone or more analytes in a bodily sample, e.g., a blood sample.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1: Sensors with Conductive Microspheres to MaximizeReagent Distribution and Electrode Surface Area

In the present example, microspheres having poly(styrene) cores with adiameter of 4.99 microns (44% of total mass) were used. Thepoly(styrene) cores were coated with 0.062 microns Ni (24% of totalmass), followed by 0.039 microns Au (32% of total mass). Particledensity was approximately 2.5 g/mL. A 2.5% (w/v) tenuous microspheresuspension was prepared in a 0.1% (w/v) Triton X-100 aqueous solution.The suspension was then deposited by pipette onto Au-coated polyesterfilm working electrodes with a series of coverages varying from 1 to 2monolayers of Au microspheres. FIG. 7 provides an example photographshowing an approximately one monolayer of microspheres deposited, asthere are minimal voids and the hexagonal close packing pattern isclearly visible in many spots.

Next, a reagent solution including FAD-dependent glucose dehydrogenase(FAD-GDH; 4 units per sensor) and an osmium complex-based redox mediator(1.5 μg per strip) was deposited over the microspheres, and themicrosphere-coated strip halves were paired with Ag/AgCl-coated counterelectrode strip halves. The Ag/AgCl was first coated with a aliphaticurethane polymer to eliminate any short circuits that could be caused bymisplaced microspheres. The assembled strips, along with control stripsnot containing Au-coated microspheres, were then tested with highcontrol solution. FIG. 8 is a graph showing the average currents (n=1 to3) for different Au-coated microsphere loadings.

As shown in FIG. 8, the peak current increases as the Au-coatedmicrosphere loading increases. At the same time, the response time(i.e., the time for the current to decrease to one half of its peakvalue) decreases. Accordingly, the microspheres achieve the desiredeffect.

Example 2: Sensors with Polymer on Ag/AgCl Counter Electrode and CarbonNanopowder on Carbon Working Electrode

In this example, the following polymer was disposed on an Ag/AgClcounter electrode of a sensor having carbon nanopowder disposed on theworking electrode to improve uniformity of reagent distribution andincrease the effective surface area of the working electrode:

First, two stock solutions were prepared: (a) 50 mg/ml of the abovepolymer in 55%/45% ethanol/water with 1 mg/mL Igepal CO-610, 5 mg/mLPluronic F-108, 1 mg/mL NaCl and 25 mM HEPES buffer and (b) 200 mg/mlpoly(ethylene glycol) diglycidyl ether (Mw=400) in a mixture of 80%ethanol and 20% 10 mM hepes buffer (pH 8.0).

The final coating solution was prepared by mixing solutions (a) and (b)in a 20:1 ratio and rocking the solution for 30 minutes. A substrateincluding an Ag/AgCl counter electrode solution was then coated with thefinal coating solution at a deposition rate of 2.4 μL/cm, followed bydrying at 80° C. under air. A clear thin film was formed on top of thecounter electrode trace. Checking with a multimeter probe indicated thatthe film insulated the underlying Ag/AgCl counter electrode. A controlAg/AgCl counter electrode was coated with 2 mg/mL Igepal CO-610, 10mg/mL Pluronic F-108, 2 mg/mL NaCl and 50 mM HEPES buffer in 90%/10%water/ethanol.

Carbon nanopowder was used as the conducting particle on a carbonworking electrode included on a second substrate. A solution containing2.5% carbon nanopowder and other standard working electrode detectionreagents (FAD-GDH enzyme (4 Unit per strip), osmium transition metalmediator (1.5 μg per strip)) was coated in the working electrodechannel. These substrates were then paired with the substrates includingthe counter electrode with and without the polymer coating, as describedabove. The combined substrates were cut to make electrochemical teststrips.

Shorting between the working and counter electrodes was checked byapplying a 100 mV voltage over the two sides of a strip on apotentiostat without applying any glucose solution to the strip channel.A current indicated the presence of shorting. The results showed thatthe strips without the polymer coating had about a 50% shorting rate,while the strips with the polymer coating disposed on the counterelectrode did not exhibit any shorting.

The glucose current profiles of the non-shorted strips from the group ofstrips without the polymer coating and the strips from the polymercoated group were compared when a glucose solution was applied. It wasfound that strips from the two groups gave indistinguishable glucosecurrent profiles, indicating that the polymer coating indeed providedthe necessary ionic conductivity.

Example 3: Sensors with Polymer on Ag/AgCl Counter Electrode and CarbonNanopowder on Gold Working Electrode

In this example, the same polymer coating was disposed on a counterelectrode to perform the same experiment as described in Example 2.Here, however, gold-coated polyester, rather than carbon, was used asthe working electrode. The results showed that the polymer coatingprevented shorting from occurring between the working and counterelectrodes.

Example 4: Sensors with Neorez R-9603 Polymer on Ag/AgCl CounterElectrode and Gold-Coated Polystyrene Microspheres on Gold WorkingElectrode

In this experiment, the conductive particles on the working electrodeside were gold-coated polystyrene microspheres (5 μm diameters). Thepolymer coating was formed using NEOREZ® R-9603 aliphatic urethanepolymer (DSM NeoResins, Waalwijk, The Netherlands). The experimentaldetails were as described above in Example 1. The larger size of thegold-coated microspheres used in this study was more likely to causeshorting between the working and counter electrodes. However, suchshorting did not occur in the strips that included the polymer coatingon the counter electrode, indicating the effectiveness of the polymercoating in preventing shorting between the working and counterelectrodes in the presence of larger conductive particles, e.g., 5 μmdiameter conductive microspheres. In addition, the strips successfullydelivered very high peak current (140 μA), indicating sufficient ionicconductivity of the polymer coating.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

The invention claimed is:
 1. A biosensor, comprising: a first substrate;a second substrate disposed over the first substrate; an electrodedisposed between the first substrate and second substrate; and a drycomposition disposed on an area of the electrode, the compositioncomprising an analyte detection reagent and electrically conductiveparticles, wherein the analyte detection reagent comprises ananalyte-responsive enzyme and the electrically conductive particles arepresent in an amount of about 10² to about 10¹⁰ conductive particles permm² of the area of the electrode.
 2. The biosensor of claim 1, whereinthe conductive particles comprise a material selected from the groupconsisting of gold, carbon, cobalt carbon, platinum, ruthenium,palladium, silver, silver chloride, silver bromide, aluminum, copper,copper-zinc, diamond, iron, iron-nickel, molybdenum, magnesium, nickel,palladium, platinum, silver-copper, tantalum, tin, indium doped tinoxide, titanium, titanium nitride, tungsten, zinc, calcium oxide,hydroxyapatite, indium, silica, silicon, silicon dioxide, siliconnitride, silicon carbide, and any combination thereof.
 3. The biosensorof claim 1, wherein the conductive particles comprise nanoparticles. 4.The biosensor of claim 3, wherein the nanoparticles have a diameter inthe range of from about 1 nm to about 300 nm.
 5. The biosensor of claim1, wherein the conductive particles comprise a carbon nanopowder.
 6. Thebiosensor of claim 1, wherein the conductive particles are microspheres.7. The biosensor of claim 6, wherein the microspheres comprise a polymercoated with a conducting material.
 8. The biosensor of claim 6, whereinthe microspheres are gold-coated polystyrene microspheres.
 9. Thebiosensor of claim 6, wherein the microspheres have a diameter in therange of from about 0.5 μm to about 100 μm.
 10. The biosensor of claim6, wherein the microspheres have an average diameter of about 5 μm. 11.The biosensor of claim 1, wherein the electrode is a working electrodeand the biosensor further comprises a counter electrode comprising apolymer layer disposed on the counter electrode.
 12. The biosensor ofclaim 1, wherein the electrode is disposed on the first substrate or onthe second substrate.
 13. The biosensor of claim 1, wherein drycomposition further comprises a redox mediator.
 14. The biosensor ofclaim 1, wherein the analyte-responsive enzyme is a glucose-responsiveenzyme.
 15. The biosensor of claim 1, is an in vivo biosensor.
 16. Amethod comprising: determining an analyte concentration of a user usinga biosensor, the biosensor comprising: a first substrate; a secondsubstrate disposed over the first substrate; an electrode disposedbetween the first substrate and second substrate; and a dry compositiondisposed on an area of the electrode, the composition comprising ananalyte detection reagent and electrically conductive particles, whereinthe analyte detection reagent comprises an analyte-responsive enzyme andthe electrically conductive particles are present in an amount of about10² to about 10¹⁰ conductive particles per mm² of the area of theelectrode.
 17. The method of claim 16, wherein the conductive particlescomprise a material selected from the group consisting of gold, carbon,cobalt carbon, platinum, ruthenium, palladium, silver, silver chloride,silver bromide, aluminum, copper, copper-zinc, diamond, iron,iron-nickel, molybdenum, magnesium, nickel, palladium, platinum,silver-copper, tantalum, tin, indium doped tin oxide, titanium, titaniumnitride, tungsten, zinc, calcium oxide, hydroxyapatite, indium, silica,silicon, silicon dioxide, silicon nitride, silicon carbide, and anycombination thereof.
 18. The method of claim 16, wherein theanalyte-responsive enzyme is a glucose-responsive enzyme.
 19. The methodof claim 16, wherein the biosensor is an in vivo biosensor.
 20. Abiosensor, comprising: a first substrate; an electrode disposed on thefirst substrate; and a dry composition disposed on an area of theelectrode, the composition comprising an analyte detection reagent andelectrically conductive particles, wherein the analyte detection reagentcomprises an analyte-responsive enzyme and the electrically conductiveparticles are present in an amount of about 10² to about 10¹⁰ conductiveparticles per mm² of the area of the electrode, and wherein theelectrically conductive particles provide for uniform distribution ofthe analyte-responsive enzyme on the area of the electrode.